The coagulation cascade, in vertebrates, has evolved to prevent the loss of blood (hemorrhaging) by the process of hemostasis.  Hemostasis is a sensitive balance between three systems, coagulation, fibrinolysis and anticoagulation (reviewed in 2).  A disruption of this balance, towards excessive coagulation, may result in thrombosis while a shift towards excessive fibrinolysis made lead to increased bleeding (reviewed in 2).   As reviewed by Davidson et al. there are five proteases (factor VII, factor IX, factor X, protein C and prothrombin)  that function along with five cofactors (Tissue Factor (TF), factor V, factor VIII, thrombomodulin and protein S) during coagulation 1.  TF, a 47-kDa protein, is a member of the cytokine receptor family and initiator of the coagulation cascade 3.  This leads to the generation of fibrin from fibrinogen through the activity of thrombin (reviewed in 1).  The effects of coagulation are reversed by fibrinolysis, which is involved in the breakdown of fibrin (reviewed in 4).  Members of the fibrinolytic cascade include tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) and plasminogen (reviewed in 4).  The activity of plasmin, from the plasminogen precursor, leads to the breakdown of fibrin into fibrin degradation products (i.e. D-dimers) (reviewed in 4).  The coagulation cascade is also regulated by the anticoagulation system.  A key component of this system is protein C which, when activated, functions primarily to inhibit the process of coagulation (reviewed in 5).  From this, it is evident that hemostasis is a tightly regulated system that is sensitive to a disruption in coagulation, fibrinolysis or anticoagulation.

Ebola virus (EBOV) is a negative sense, single-stranded, nonsegmented, RNA virus from the Filoviridae family (reviewed in 6) that first emerged in 1976 7; 8.  EBOV, the causative agent of Ebola Hemorrhagic Fever (EHF), is highly pathogenic to both humans and nonhuman primates 6.  Due to this extreme pathogenicity and high mortality rate, EBO is classified as a Biosafety Level 4 agent by the Center for Disease Control and Prevention (CDC) and therefore, many studies are conducted using animal models such as guinea pigs, baboons and rhesus monkeys 9.  It is interesting to note that despite twenty-eight years of research and numerous tests of various species, a natural reservoir has not been identified for EBOV.

            As mentioned previously, infection with EBOV causes a lethal syndrome known as viral hemorrhagic fever 6.  Unfortunately, there is no vaccine or antiviral therapy for this infectious disease despite numerous attempts to inhibit viral replication 10.  Infected individuals suffer from a multitude of symptoms including fever, chills, anorexia, dizziness and myalgia.  Symptoms of severe cases include nausea, abdominal pain, diarrhea, vomiting, and excessive bleeding from internal organs, orifices and under the skin.  Surprisingly, patients do not succumb to blood loss, but rather shock, seizures, coma and nervous system malfunction 6.

            Dysregulation of the coagulation cascade resulting in disseminated intravascular coagulation (DIC) is another symptom of EHF 7; 11; 12.  DIC has been described in various reviews as the localized or systemic activation of the coagulation cascade resulting in histologically visible microthrombi in the microvasculature 13; 14.  Geisbert et al. suggest that the multiple organ failure from an EBOV infection is the result of microthrombi impeding blood flow to the organ(s) 15.  Interestingly, Mammen outlines that TF, in most instances, is responsible for initiating DIC 14 and therefore, plays an important role in the progress of this symptom.

            A 2003 report characterizing the mechanism of coagulation anomalies (i.e. DIC) during the course of an EBOV infection, in non-human primates, demonstrated that TF was overexpressed by both monocytes and macrophages.  This overexpression was demonstrated by in vivo studies that found TF mRNA transcripts increased in the peripheral blood mononuclear cells (PBMC) of rhesus macaques during the course of EBOV infection 15 and from immunofluorescence assays (IFA) detecting an increase in TF protein expression in PBMC 15.  These findings suggest a potential role for TF in EBOV pathogenicity.  

            The in vivo findings of increased TF expression in EBOV infected rhesus macaques were corroborated by in vitro studies that demonstrated primary human monocytes/macrophages (PHMs) infected with EBOV immediately, 1-hour post infection, upregulated TF mRNA transcripts 15.  Furthermore, EBOV infected PHMs also demonstrated increased TF expression as determined by IFA 15.  These studies not only confirm the in vivo findings of TF overexpression, but together, they suggest a pivotal role for TF during the course of an EBOV infection.          

            Interestingly Geisbert et al. demonstrated, both in vivo and in vitro, an increase in microparticles (MPs) expressing TF during the course of an EBOV infection.  Immunoelectron microscopy (IEM) studies of fluid from PHM cultures detected TF-positive MPs by gold-sphere labeling for TF while this same technique also detected TF-positive MPs in plasma from EBOV infected rhesus macaques 15.  TF-positive MPs have previously been shown to increase both the generation of thrombin, in vitro, and the risk of thromboembolic events 16; 17.

            Based on the previous findings of TF overexpression in monocytes and macrophages of EBOV infected primates, Geisbert et al. hypothesized that the TF-pathway exacerbates the effects of EHF and therefore, these pathological effects could be improved by TF-pathway inhibition 18.  The group tested this hypothesis by using a recombinant nematode anticoagulant protein c2 (rNAPc2) which binds to either factor X or Xa and directly inhibits the TF/factor VIIa complex (reviewed in 19).  Surprisingly, treatment of rhesus monkeys with rNAPc2 prolonged survival when challenged with a lethal dose of EBOV and, 33% of treated monkeys survived the infection 18(reviewed in 20).  Furthermore, the survivors showed reduced TF activity as measured by a fluorogenic cleavage assay 18.  Therefore, these findings suggest a potential therapy to target the disease process rather than the conventional approach of targeting viral replication for EBOV infections 18.

            As reviewed by both Mammen and Levi, DIC results in various coagulation abnormalities of procoagulant, anticoagulant and fibrinolytic factors 13; 14.  For example, a 2001 study found that both the activated partial thromboplastin (aPTT) and the prothrombin (PT) times were prolonged in a macaque that succumbed to a lethal EBOV challenge 21.  Also, Geisbert and colleagues found that protein C levels decreased by day 2 in macaques challenged with EBOV and remained low throughout the course of infection 15 (reviewed in 22).  Furthermore, these studies in macaques demonstrated that both tPA and uPA levels increased by day 4, post-infection, and continued to do so throughout the course of infection 15; 22.  Finally two unrelated studies that characterized the development of fibrin-degradation products (D-dimers) in the plasma of macaques, found the results to be consistent with DIC.  In all macaques that succumbed to EBOV infection, there was a rapid increase in D-dimers by day 5 15; 21; 22.  Interestingly, two macaques that survived the lethal challenge developed D-dimers more slowly, peaking by day 14 15; 21; 22.  Taken together, these results clearly demonstrate that there is a dysregulation of both anticoagulant and fibrinolytic factors during the course of EBOV infection in macaques.   

            As mentioned previously, many studies on the effect of EBOV on hemostatis stem from the use of animal models.  This leads to the question, are these animal models indicative of the effect of EBOV on hemostasis in humans?  Surprisingly, Ryabchikova and colleagues have shown that non-human primates exhibit differences in hemostasis when challenged with EBOV.  Green monkeys were found to undergo fibrin thrombosis, which was in sharp contrast to baboons, which exhibited hemorrhagic symptoms 23.  Furthermore, previous findings from this group demonstrated that guinea pigs are an unsuitable model because they did not show any signs of hemorrhagic syndrome 24.  These findings, therefore, suggest species-specific effect on hemostasis, which may not be representative of human EBOV infection.    

            Hemostasis is a delicate and well-balanced process necessary to avoid such pathological processes as hemorrhaging.  EHF is an infectious disease in which the balance of hemostasis is disrupted resulting is DIC 7; 11; 12.  Infection with EBOV results in the dysregulation of not only procoagulant factors, but also factors of both the fibrinolytic and anticoagulant systems 15; 18; 21.  TF has recently been identified as a key player in the disease process and was found to be overexpressed in both in vitro and in vivo studies 15.  Furthermore, therapeutic treatment of macaques with rNAPc2 was found to increase the mean survival and to reduce the coagulation response following a lethal challenge of EBOV 18.  Various procoagulant (TF) 15; 18, anticoagulant (protein C) 15; 18 and fibrinolytic (tPA and uPA) 15 factors were also found to be dysregulated during the course of an EBOV infection in macaques.  Based on this, it is clear that EBOV disrupts coagulation, anticoagulation and fibrinolysis, but, potentially, can be treated by TF-inhibition 18.  Further studies should therefore aim to not only expand on the benefits of TF-inhibitors, but should also be combined with anticoagulant therapy (i.e. recombinant activated protein C) for a synergistic regimen to treat EBOV infections.    

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